The Etiology of Bloodstream Infections at an Italian Pediatric Tertiary Care Hospital: A 17-Year-Long Series

Knowledge of epidemiology is essential for guiding correct antibiotic prescription, reducing bacteremia-associated mortality, and implementing targeted infection control programs. However, only a few studies have reported on the epidemiology of bloodstream infections (BSIs) in pediatrics. We performed a retrospective analysis of all BSIs (excluding those caused by common skin contaminants) diagnosed from 2006 to 2022 in patients younger than 18 years who were treated at an Italian pediatric tertiary care hospital. Overall, 2395 BSIs were recorded, including 2207 (92.15%) due to bacteria and 188 (7.85%) due to fungi. The incidence rate (BSIs/10,000 hospital discharges, IR) of bacterial BSIs significantly increased during the study period. In particular, BSIs caused by S. aureus (including MRSA), Enterobacterales (including ESBL and AmpC producers), Enterococcus spp., and P. aeruginosa became more common. The frequency of carbapenem-resistant strains was <1% and stable over time. Conversely, there was a significant reduction in the incidence of BSIs due to S. pneumoniae. The BSIs were stratified by patient age, and S. aureus was the most frequent cause of BSIs in all age groups, while E. coli was the most frequent in the Enterobacterales family. S. agalactiae was the third most frequent cause of neonatal early-onset BSIs. The prevalence of Enterococcus spp. increased in the subgroups from 8 days to 5 years of age, while P. aeruginosa became more prevalent in children over 5 years of age. S. aureus was also the most frequent isolate in both community- and hospital-onset BSIs, followed by E. coli. The prevalence of multidrug-resistant (MDR) pathogens was very low. It was <5% for both Gram-positive (i.e., MRSA and VRE) and Gram-negative (ESBL, AmpC, and carbapenem-resistant) pathogens, and MDR pathogens were almost exclusively detected in hospital-onset BSIs. Fungi accounted for just under 8% of BSIs. C. albicans was the most frequently isolated strain, followed by C. parapsilosis. Notably, the IR of fungemia did not change significantly during the study period, in spite of an increase in the absolute number of events. The continuous monitoring of local epidemiology is essential to identify changes in the IRs of pathogens and antibiotic susceptibility and to guide antibiotic treatments, especially in the phase when antibiograms are not yet available.


Introduction
Bloodstream infections (BSIs) significantly contribute to pediatric morbidity and mortality, particularly when they trigger sepsis and septic shock.In 2017, approximately 25 million children worldwide developed sepsis, resulting in over 3 million deaths [1].In children, guidelines recommend starting antimicrobial therapy within 3 h after the diagnosis of sepsis, and even within 1 h in cases of septic shock [2], since the prompt and appropriate treatment of BSIs is crucial to prevent progression to septic shock and death.However, the identification of the causative agent of a BSI and the determination of its antibiotic susceptibility may take 48-72 h, thus making it necessary to carry out empirical antimicrobial therapy based on local epidemiology, also considering factors such as age, sex, and the presence of comorbidities [2].In the era of antimicrobial resistance (AMR) [3,4], knowledge of local epidemiology is essential for guiding empirical therapy, especially in critically ill patients who may require broad-spectrum coverage that includes multidrug-resistant (MDR) organisms [5,6].AMR prevalence varies over time, in different geographical regions, and even among units within the same hospital [7].General epidemiology and AMR rates can change over time due to the introduction of vaccines, peripartum/surgical prophylaxis, pandemics (e.g., SARS-CoV-2), antibiotic prescription practices, and infection control measures [8].Despite the recognized importance of epidemiology in guiding antibiotic prescription [9], few studies have reported on the epidemiology of BSIs in pediatric settings [10][11][12].
This study aims to describe the etiology of BSIs in children hospitalized at a tertiary care pediatric hospital in northern Italy, focusing on the age distribution, community-or hospital-onset, and major susceptibility patterns, while also analyzing epidemiological variations over time.

Materials and Methods
This study presents a retrospective review of all positive blood cultures collected from children admitted to the IRCCS Istituto Giannina Gaslini (IGG), Genoa, Italy, from 1 January 2006 to 31 December 2022.The IGG is a tertiary care pediatric hospital in northern Italy that serves as a local pediatric hospital and a referral hospital nationwide and for many foreign countries.
BSIs diagnosed at admission or during hospitalization in patients aged 0-18 years were included in this study.Episodes were identified in a microbiology database.The only demographic variable, collected anonymously, was the age at BSI diagnosis.Additionally, the ward where blood cultures were sampled was recorded.A BSI was defined by the isolation of a pathogenic microorganism from at least one aerobic/anaerobic set of blood culture bottles.For bacterial BSIs (bacteremias), blood cultures growing common skin contaminants [13] were excluded from the analysis.According to the literature, contaminants were defined as microorganisms that are thought to be introduced to cultures during specimen collection or processing and are not generally pathogenic for patients [14].Microorganisms with contamination rates ≥ 50% were considered contaminants [14][15][16][17] (see Supplementary Table S1).Blood cultures positive for fungi (fungemias) were included.The patients could have repeated positive blood cultures: cultures from an individual patient that were positive for the same pathogens within two weeks of the initial isolate were considered a single incident.
Among the bacteremias, methicillin-resistant Staphylococcus aureus (MRSA) were defined as S. aureus resistant to oxacillin, and vancomycin-resistant Enterococcus spp.(VRE) were defined as Enterococcus spp.resistant to vancomycin.Among the Gramnegative pathogens, due to the occasional unavailability of molecular tests to differentiate between extended-spectrum β-lactamases (ESBLs) and AmpC throughout the study period, ESBL-producing microorganisms were pragmatically defined as those resistant to third-generation cephalosporins (cefotaxime and/or ceftazidime) and a fourthgeneration cephalosporin (cefepime) (third/fourth generation cephalosporins, 3/4GC).AmpC-producing microorganisms were defined as those resistant to a third-generation cephalosporins but susceptible to cefepime.Carbapenem resistance was defined as resistance to meropenem.Resistance to specific antibiotics was defined based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines, which classify microorganisms as follows: susceptible (S), when there is a high likelihood of therapeutic success using a standard dosing regimen; susceptible increased exposure (I), when there is a likelihood of therapeutic success if exposure to the agent is increased by adjusting the dosing regimen or by its concentration at the site of infection; resistant (R), when there is a high likelihood of therapeutic failure even with increased exposure [18].The category "I" was combined with "S" since the hospital policy typically administers the maximal doses of antibiotics.Interpretations were used instead of the minimal inhibitory concentration (MIC) values due to the potential changes in the EUCAST interpretation of MIC over the years.
A BSI was categorized as community-onset if the positive blood culture was performed in the emergency or short-stay observation ward; it was categorized as hospital-onset if the blood culture was collected in any other hospital wards.

Statistical Analysis
A descriptive analysis was conducted for the distribution of different pathogens across the entire patient population, stratified by age group and the ward where the blood culture was sampled.Descriptive analyses were used to explore the demographic factors of patients with BSIs (i.e., age and ward at the time of the BSI) and to summarize the bacterial isolates and anti-microbial susceptibility patterns.The annual incidence rate (IR) of BSIs was calculated as the ratio between the number of BSIs diagnosed over one year and the number of hospital discharges in that year, normalized to 10,000 discharges.The Spearman test was used to evaluate changes in the IR during the study period (Social Science statistics https://www.socscistatistics.com/).Two-sided p values < 0.05 were considered statistically significant.

Age Group Stratification
Table 3 reports the frequencies of the most representative microorganisms stratified by age group.S. aureus was among the top three pathogens in all age subgroups, while S. agalactiae was the third most frequent cause of neonatal early-onset BSI.Among the Gram-negative pathogens, E. coli was the most representative pathogen across all the age subgroups.The prevalence of Enterococcus spp.increased in neonates and infants, while P. aeruginosa became more prevalent in children.C. albicans was mainly detected in the first month of life, while C. parapsilosis was more frequently isolated in older patients.

Community-vs. Hospital-Onset Infections
Table 4 reports the etiologies of BSIs classified as community-or hospital-onset.In the community-onset infections, S. aureus was the most frequently isolated pathogen, followed by E. coli, S. pneumoniae, and S. agalactiae.Out of the hospital-onset infections, S. aureus was again the most frequent isolate, followed by E. coli, E. faecalis, and K. pneumoniae.Interestingly, the proportions of S. pyogenes BSIs were quite similar in the communityand hospital-onset infections (60% (n = 12/20) and 40% (n = 8/20), respectively).Notably, only a few MRSA-(6/97 and 6.18%), ESBL-(2/94 and 2.13), and AmpC-(2/67, 2.98%) producing Enterobacterales were isolated in the community-onset BSIs.Fungemia was nearly exclusively diagnosed in the hospital-onset infections, with Candida albicans representing about 3% of all the identified causes of BSI.

Incidence Rates and Trends over Time
Figure 1 illustrates the overall IRs of bacteremias and fungemias and their trends over time.A statistically significant increase in the bacteremia IR was observed (rs = 0.85454, p = 0.00081), whereas no significant change was observed for fungemias (rs = 0.01226, p = 0.96277).Table 5 reports the IRs of different causes of BSIs over the years.Statistically significant increases were found in the BSIs due to S. aureus (including MRSA), Enterobacterales (including ESBL and AmpC producers), Enterococcus spp., and P. aeruginosa.In contrast, there was no significant change in the IRs of VRE, meropenem-resistant Enterobacterales, or P. aeruginosa resistant to 3/4GC, piperacillin/tazobactam, or meropenem.Additionally, statistically significant reductions were observed in the IRs of BSIs due to S. pneumoniae.

Discussion
In this study, we have reported the epidemiology of BSIs diagnosed at a pediatric Italian tertiary care center from 2006 to 2022.Despite being based on data from a single center, the scarcity of pediatric data on BSI etiology and the long time span covered by study make these results important.
Unlike other studies that have reported stable BSI hospitalization rates [19], we observed a significant increase in the bacteremia IR, while the increase in the absolute number of fungemias did not result in a significant change in the IR.Enterobacterales was the most frequently identified pathogen family, with E. coli being the most prevalent.S. aureus., which was the most frequent isolate in absolute terms, showed an increasing IR over the years, along with Enterococcus spp., S. agalactiae, Enterobacterales, and P. aeruginosa.Other authors reported different trends for Enterococcus spp.and S.pyogenes [19].Notably, in our series, S.pyogenes only represented less than 1% of all BSI cases.The low IR of BSIs due to H. influenzae, S. pneumoniae, and N. meningitidis likely reflects the impact of vaccines introduced in Italy between 1995 and 2001 [20][21][22][23].While the IR of BSIs due to H. influenzae and N. meningitidis remained low and stable, the IR of BSIs due to S. pneumoniae significantly decreased.This reduction, in a region with a vaccination rate of >90% [24], is likely due to the introduction of the 13-valend pneumococcal-conjugate vaccine (PCV13), which started to substitute the PCV7 vaccine in 2010 [19].
Stratification by age showed that S. aureus was the most frequent cause of BSIs across all age groups, followed by E. coli.S. agalactiae was significant in neonates aged ≤ 7 days, as previously observed [25,26], while E. faecalis and P. aeruginosa were more prevalent in older children.Other studies have indicated a higher prevalence of S. pneumoniae and Salmonella spp.[12], contrasting with our findings of a higher prevalence of E. faecalis and P. aeruginosa.Fungi were a rare cause of BSI, accounting for just under 8%, with C. albicans and C. parapsilosis being the two most represented yeasts.This low prevalence aligns with other recent reports [27].An important limitation of our study is the exclusion of coagulasenegative staphylococci (CoNS) from the analysis.CoNS are often considered pathogens causing late-onset sepsis in low-birth weight infants, a population where contaminants could play a significant role in the epidemiology.In other age groups, CoNS may be considered pathogens in cases of central line-associated bloodstream infection (CLABSI).However, since other diagnoses were not included and this was a laboratory-based study, it is impossible to know if a central line was present at the time of blood culture.Since CLABSIs were not the main focus of this study and the presence of the line could not be determined, it is not possible to ascertain if isolates considered contaminants could be true pathogens.
When comparing community-vs.hospital-onset BSIs, S. aureus was more frequent in community-onset infections, followed by E. coli.In this setting, S. pneumoniae and S. agalactiae were also common.In contrast, in hospital-onset BSIs, S. aureus and E. coli remained frequent, with a significant presence of E. faecalis and K. pneumoniae.Fungemia was found almost exclusively in the hospital-onset BSIs.
Finally, regarding AMR pathogens, ESKAPE pathogens accounted for 20% of the BSIs, consistent with other studies [28].MRSA represented nearly 20% of the S. aureus strains, but its proportion remained < 10% across all age groups, with a slight prevalence in the hospitalonset BSIs.VRE were rare, while ESBL-and AmpC-producing Enterobacterales showed increasing trends.No significant changes were observed in carbapenemase-producing Enterobacterales or MDR P. aeruginosa.The low prevalence of MDR pathogens at our institute, which differs from observations at other centers and throughout Italy [29,30], could be attributed to the strict screening and isolation protocols adopted by our hospital [31].However, it is important to note that the data from 2020-2022 could have been influenced by the widespread reduction in MDR hospital pathogens that occurred during the SARS-CoV-2 pandemic [32].Additionally, we did not study the epidemiology in 2023, when there might have been an increase in MDR microorganisms due to increased antibiotic prescription during the pandemic [33,34].We also identified an increase in S. aureus MIC for vancomycin [35], and a concerning number of cefiderocol-resistant Gram-negative pathogens [36].To further underscore the association between MDR pathogens and hospitalization/antibiotic exposure, we observed that almost all MRSA, VRE, MDR Enterobacterales, and P. aeruginosa were responsible for hospital-onset BSIs, only a few MRSA-, ESBL-, and AmpC-producing Enterobacterales were isolated in the community-onset BSIs.
In conclusion, understanding local epidemiology is essential for guiding empirical antibiotic treatment, as the incidence of causative pathogens can change over time and among geographical areas.Despite the low proportion of MDR pathogens identified in our series, the high percentage of ESKAPE pathogens and the increases in the IRs of some resistant pathogens underscore the necessity for continued screening and infection control measures, along with prudent use of antibiotics and accurate and up-to-date diagnostic testing.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/pathogens13080675/s1.Table S1.Blood culture's contaminant microorganism.References [14][15][16][17] are cited also in Supplementary Materials.Institutional Review Board Statement: This study did not need Internal Review Board approval, as it was a purely observational retrospective study on routinely collected anonymous data.
Informed Consent Statement: Informed consent to participate in the study was waived, since retrospective data were obtained from an anonymous microbiology database.In any case, consent to completely anonymous use of clinical data for research/epidemiological purposes was requested by clinical routine at the time of admission/diagnostic procedure.

Figure 1 .
Figure 1.Incidence rate of bacteremias and fungemias by year of observation.Legend: BSI, bloodstream.

Figure 1 .
Figure 1.Incidence rate of bacteremias and fungemias by year of observation.Legend: BSI, bloodstream.

Table 1 .
Absolute numbers and proportions of different pathogens causing bloodstream infections during the study period.

Table 2 .
Absolute numbers and proportions of antibiotic-resistant organisms.

Table 3 .
Absolute numbers and proportions of pathogens isolated from bloodstream infections, stratified by age subgroups.

Table 4 .
Absolute numbers and proportions of pathogens isolated from bloodstream infections classified as community-or hospital-onset.

Table 5 .
Absolute number s and incidence rates per 10,000 admissions of pathogens isolated from bloodstream infections from 2006 to 2022.